Generation of protective vaccine responses, governed by the successful generation of T follicular helper cells and long-lived memory T cells, is increasingly impaired with age. Since microRNAs are a major regulator of the T cell proteome, we hypothesized that age-associated changes in the expression of microRNAs contribute to the defects that are seen with T cell aging. MicroRNAs are known to concomitantly reduce expression of many target molecules, frequently belonging to specific functional modules. While the effect on each of these molecules is generally small, the concerted activity on signaling and transcription factor networks can have major effects. MicroRNAs that are known to be are dynamically regulated during T cell differentiation include miR-181a and miR-21. While miR-181a expression declines with T cell differentiation, miR-21 shows the opposite pattern being higher in effector than in nave CD4 T cells. For both miRNAs, nave CD4 T cells from older individuals reflect a state of higher differentiation with decrease in miR181a and increase in miR21 expression. miR-181a is known as a rheostat of T cell receptor signaling thresholds, and indeed older nave T cells are less responsive to stimulation due to the loss of miR-181a and the associated overexpression of dual specific phosphatase 6. In preliminary studies, we have shown that miR-21 selects against T follicular helper cell differentiation. The current proposal aims at identifying the pathways controlled by these microRNAs with the ultimate goal to either target these microRNAs or the pathways that they regulate to improve immune memory in older individuals after vaccination. In Aim 1, we propose to examine how the shift in miR-181a and miR-21 expression with age selects against the development of transcriptional signatures characteristic of T follicular helper cells and T memory cells. Specifically, we will examine how these microRNAs influence transcription factor networks including FOXO, AP1, BLIMP, BCL6 and TCF1 that are important for T follicular helper and T memory cell differentiation. In Aim 2, we examine consequences of reduced miR-181a expression on the transcription of histone genes and determine the consequences of reduced histones on the nucleosome organization of effector and memory T cells and their ability to proliferate and survive. Aim 3 will examine whether transcription of pri-miR-21 and/or pri-miR-181a can be targeted to improve T cell responses. Based on preliminary studies on transcriptional regulation of these pri-miRNAs, activation of WNT signaling and inhibition of AP1 activity emerge as candidate interventions to restore miR-181a1 and reduce miR-21 expression.